title: The structure of Escherichia coli ExoIX--implications for DNA binding and catalysis in flap endonucleases.
abstract: Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme-DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK and Department of Infection & Immunity, Krebs Institute, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Anstey-Gilbert
authors.forename: Glyn R
authors.initials: GR
authors.lastname: Hemsworth
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Flemming
authors.forename: Michael R G
authors.initials: MR
authors.lastname: Hodskinson
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Svetlana E
authors.initials: SE
authors.lastname: Sedelnikova
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Stillman
authors.forename: Jon R
authors.initials: JR
authors.lastname: Sayers
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Artymiuk
mesh_terms: D055162:Biocatalysis; D002118:Calcium / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011188:Potassium / Q000737:chemistry
chemical_list: D004247:DNA; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C113094:exonuclease IX, E coli; C468317:FEN1 protein, human; D010727:Phosphoric Diester Hydrolases; D008274:Magnesium; D011188:Potassium; D002118:Calcium